Bacillus thuringiensis strains and their insecticidal proteins

ABSTRACT

Four novel  Bacillus thuringiensis  strains, which are deposited at the BCCM-LMG under accession nos. LMG P-12592, LMG P-12593, LMG P-12594, and LMG P-13493, produce new crystal proteins during sporulation that are toxic to Lepidoptera, more particularly against Noctuidae such as Spodoptera spp. and  Agrotis ipsilon , against Pyralidae such as  Ostrinta nubilalis , and against Yponomeutidae such as  Plutella xylostella , and that are encoded by a novel gene. The crystal proteins contain protoxins, which can yield a toxin as trypsin-digestion product. A plant, the genome of which is transformed with a DNA sequence that comes from either one of the strains and that encodes its respective toxin, is resistant to Lepidoptera. Each strain, itself, or its crystals, crystal proteins, protoxin or toxin can be used as the active ingredient in an insecticidal composition for combatting Lepidoptera.

This application is a division of U.S. application Ser. No. 08/379,656,filed Mar. 23, 1995, now U.S. Pat. No. 5,885,571 which is a nationalstage filing of PCT/EP93/01820 filed Jul. 12, 1993.

This invention relates to four novel strains of Bacillus thuringiensis(the “BTS02617A strain”, the “BTS02618A strain”, the “BTS02654B strain”and the “BTS02652E strain”), each of which produces crystallizedproteins (the “BTS02617A crystal proteins”, the “BTS02618A crystalproteins”, the “BTS02654B crystal proteins” and the “BTS02652E crystalproteins”, respectively) which are packaged in crystals (the “BTS02617Acrystals”, the “BTS02618A crystals”, the “BTS02654B crystals” and the“BTS02652E crystals”, respectively) during sporulation. The BTS02617A,BTS02618A, BTS02654B and BTS02652E strains were deposited under theprovisions of the Budapest Treaty at the Belgian Coordinated Collectionsof Microorganisms—Collection Laboratorium voor Microbiologie Belgium(“BCCM-LMG”), R.U.G., K. Ledeganckstraat 35, B-9000 Gent.

This invention also relates to an insecticide composition that is activeagainst Lepidoptera and that comprises the BTS02617A, BTS02618A,BTS02654B or BTS02652E strain, as such, or preferably the BTS02617A,BTS02618A, BTS02654B or BTS02652E crystals, crystal proteins or theactive component(s) thereof as an active ingredient.

This invention further relates to a gene (the “bTS02618A gene”), whichis present in the genome of the BTS02617A, BTS02618A, BTS02654B andBTS02652E strains and which encodes an insecticidal protein (the“BTS02618A protoxin”) that is found in the BTS02617A, BTS02618A,BTS02654B and BTS02652E crystals. The BTS02618A protoxin is the proteinthat is produced by the BTS02617A, BTS02618A, BTS02654B and BTS02652Estrains before being packaged into their respective BTS02617A,BTS02618A, BTS02654B and BTS02652E crystals.

This invention still further relates to a toxin (the “BTS02618A toxin”)which can be obtained (e.g., by trypsin digestion) from the BTS02618Aprotoxin. The BTS02618A toxin is an insecticidally active protein whichcan be liberated from the BTS02617A crystals, the BTS02618A crystals,the BTS02654B crystals, and the BTS02652E crystals, which are producedby the BTS02617A strain, the BTS02618A strain, the BTS02654B strain andthe BTS02652E strain, respectively. This toxin and its protoxin have ahigh activity against a wide range of lepidopteran insects, particularlyagainst Noctuidae, especially against Spodoptera and Agrotis spp., butalso against other important lepidopteran insects such as Pyralidae,particularly the European corn borer, Ostrinia nubilalis, andYponomeutidae such as Plutella xylostella. This new characteristic ofthe BTS02618A protoxin and toxin (“(pro)toxin”), i.e., the combinationof activity against different economically important Lepidopteran insectfamilies such as Noctuidae, Yponomeutidae and Pyralidae, makes this(pro)toxin an ideally suited compound for combatting a wide range ofinsect pests by contacting these insects with the (pro)toxin, e.g., byspraying or by expressing the bTS02618A gene in plant-associatedbacteria or in plants. The BTS02618A toxin is believed to represent thesmallest portion of the BTS02618A protoxin which is insecticidallyeffective against Lepidoptera.

This invention yet further relates to a chimeric gene that can be usedto transform a plant cell and that contains the following operablylinked DNA fragments:

1) a part of the bTS02618A gene (the “insecticidally effective bTS02618Agene part”) encoding an insecticidally effective portion of theBTS02618A protoxin, preferably a truncated part of the bTS02618A gene(the “truncated bTS02618A gene”) encoding just the BTS02618A toxin;

2) a promoter suitable for transcription of the insecticidally effectivebTS02618A gene part in a plant cell; and

3) suitable 3′ end transcript formation and polyadenylation signals forexpressing the insecticidally effective bTS02618A gene part in a plantcell.

This chimeric gene is hereinafter generally referred to as the“bTS02618A chimeric gene”.

This invention also relates to:

1) a cell (the “transformed plant cell”) of a plant, such as corn orcotton, the genome of which is transformed with the insecticidallyeffective bTS02618A gene part, preferably the bTS02618A chimeric gene;and

2) a plant (the “transformed plant”) which is regenerated from thetransformed plant cell or is produced from the so-regenerated plant andtheir seeds, the genome of which contains the insecticidally effectivebTS02618A gene part, preferably the bTS02618A chimeric gene, and whichis resistant to Lepidoptera.

This invention still further relates to

1) a microbial organism, such as B. thuringiesis or Pseudomonas spp.,the genome of which is transformed with all or part of the bTS02618Agene; and

2) a microbial spore, containing a genome which is transformed with allor parts of the bTS02618A gene.

BACKGROUND OF THE INVENTION

B. thuringiesis (“Bt”) is a Gram-positive bacterium which producesendogenous crystals upon sporulation. The crystals are composed ofproteins which are specifically toxic against insect larvae. Thesecrystal proteins and corresponding genes have been classified based ontheir structure and insecticidal spectrum (Höfte and Whiteley, 1989).The four major classes are Lepidoptera-specific (cryI), Lepidoptera- andDiptera-specific (cryII), Coleoptera-specific (cryIII), andDiptera-specific (cryIV) genes.

The fact that conventional submerged fermentation techniques can be usedto produce Bt spores on a large scale makes Bt bacteria commerciallyattractive as a source of insecticidal compositions.

Gene fragments from some Bt strains, encoding insecticidal proteins,have heretofore been identified and integrated into plant genomes inorder to render the plants insect-resistant. However, obtainingexpression of such Bt gene fragments in plants is not a straightforwardprocess. In order to achieve optimal expression of an insecticidalprotein in plant cells, it has been found necessary to engineer each Btgene fragment in a specific way so that it encodes a part of a Btprotoxin that retains substantial toxicity against its target insects(European patent application (“EPA”) 86/300,291.1 and 88/402,115.5; U.S.patent application Ser. No. 821,582, filed Jan. 22, 1986).

SUMMARY OF THE INVENTION

In accordance with this invention, four novel Bt strains, i.e., theBTS02617A, BTS02618A, BTS02654B and BTS02652E strains, are provided. TheBTS02617A, BTS02618A, BTS02654B and BTS02652E crystals and crystalproteins, the BTS02618A protoxin and toxin produced by the strainsduring sporulation, and insecticidally effective portions of theBTS02618A protoxin, as well as equivalents of these crystals, crystalproteins, protoxin, toxin and insecticidally effective protoxinportions, each possess insecticidal activity and can therefore beformulated into insecticidal compositions against Lepidoptera ingeneral, and particularly against Noctuidae, such as Agrotis spp.(cutworms such as Agrotis ipsilon), Mamestra spp. (e.g., the cabbagemoth, Mamestra brassica) and Spodoptera spp. (armyworms, such asSpodoptera exigua, Spodoptera frugiperda, Spodoptera littoralis andSpodoptera litura), against Pyralidae (e.g., the European corn borer,Ostrinia nubilalis) and Yponomeutidae (such as Plutella xylostella)which are major pests of various economically important crops, such ascorn, cotton and many vegetables such as Brassicas.

Also in accordance with this invention, a plant cell genome istransformed with the insecticidally effective bTS02618A gene part,preferably the truncated bTS02618A gene, or an equivalent thereof suchas a modified, synthetic bTS02618A gene. It is preferred that thistransformation be carried out with the bTS02618A chimeric gene. Theresulting transformed plant cell can be used to produce transformedplants, seeds of transformed plants and plant cell cultures consistingessentially of the transformed cells. The transformed cells in some orall of the tissues of the transformed plants: 1) contain theinsecticidally effective bTS02618A gene part as a stable insert in theirgenome, and 2) express the insecticidally effective bTS02618A gene partby producing an insecticidally effective portion of its BTS02618Aprotoxin, preferably its BTS02618A toxin, thereby rendering the plantresistant to Lepidoptera. The transformed plant cells of this inventioncan also be used to produce, for recovery, such insecticidal Btproteins.

Further in accordance with this invention, a process is provided forrendering a plant resistant to Lepidoptera by transforming the plantcell genome with the insecticidally effective bTS02618A. gene part,preferably the truncated bTS02618A gene, or an equivalent thereof. Inthis regard, it is preferred that the plant cell be transformed with thebTS02618A chimeric gene.

Yet further in accordance with this invention, there are provided theBTS02618A protoxin, the insecticidally effective portions of suchprotoxin and the BTS02618A toxin, as well as functional parts of theBTS02618A toxin, as well as the bTS02618A gene, the insecticidallyeffective bTS02618A gene part, the truncated bTS02618A gene and thechimeric bTS02618A gene, as well as their equivalents.

Also in accordance with this invention, a DNA sequence, either naturalor artificial, encoding the BTS02618A protoxin or insecticidallyeffective portions thereof, such as the toxin, is provided.

Also in accordance with this invention are provided an insecticidalcomposition against Lepidoptera, particularly Noctuidae, Pyralidae andYponomeutidae, and a method for controlling Lepidoptera, particularlyNoctuidae, Pyralidae and Yponomeutidae, with the insecticidalcomposition, wherein the insecticidal composition comprises theBTS02617A, BTS02618A, BTS02654B or BTS02652E strain, crystals and/orcrystal proteins or the BTS02618A protoxin, toxin and/or insecticidallyeffective protoxin portions or their equivalents.

DETAILED DESCRIPTION OF THE INVENTION

The BTS02618A protoxin of this invention can be isolated in aconventional manner from the BTS02617A strain, deposited on July, 2 atthe BCCM-LMG under accession number LMG P-12592, the BTS02618A strain,deposited on Jul. 2, 1992 at the BCCM-LMG under accession number LMGP-12593, the BTS02654B strain, deposited on Jul. 2, 1992 at the BCCM-LMGunder accession number LMG P-12594, or the BTS02652E strain deposited onMar. 1, 1993 at the BCCM-LMG under accession number LMG P-13493. Forexample, the BTS02617A, BTS02618A, BTS02654B or BTS02652E crystals canbe isolated from sporulated cultures of their respective strain(Mahillon and Delcour, 1984), and then, the BTS02618A protoxin can beisolated from the crystals according to the method of Höfte et al.(1986). The protoxins can be used to prepare monoclonal or polyclonalantibodies specific for the protoxin in a conventional manner (Höfte etal., 1988). The BTS02618A toxin can be obtained by protease (e.g.,trypsin) digestion of the BTS02618A protoxin.

The bTS02618A gene can be isolated in a conventional manner. ThebTS02618A gene can be identified in the BTS02617A, BTS02618A, BTS02654Bor BTS02652E strain, using the procedure described in U.S. patentapplication Ser. No. 821,582, filed Jan. 22, 1986, and in EPA86/300,291.1 and 88/402,115.5 (which are incorporated herein byreference). The bTS02618A gene was identified by: digesting total DNAfrom one of the above strains with restriction enzymes; sizefractionating the DNA fragments, so produced, into DNA fractions of 5 to10 Kb; ligating these fractions to cloning vectors; screening the E.coli, transformed with the cloning vectors, with a DNA probe that wasconstructed from a region of the cryIG gene (Smulevitch et al., 1991;Gleave et al., 1992).

The term “bTS02618A gene” as used herein includes a DNA sequenceencoding the BTS02618A protoxin or toxin or functionally equivalentvariants thereof. Indeed, because of the degeneracy of the genetic code,some amino acid codons can be replaced with others without changing theamino acid sequence of the protein. Furthermore, some amino acids can besubstituted by other equivalent amino acids without significantlychanging the insecticidal activity of the protein. Also, changes inamino acid composition in regions of the molecule, different from thoseresponsible for binding and toxicity are less likely to cause adifference in insecticidal activity of the protein. Such equivalents ofthe gene include DNA sequences hybridizing to the DNA sequence of theBTS02618A toxin or protoxin of SEQ ID. No. 4 and encoding a protein withthe same insecticidal characteristics as the BTS02618A (pro)toxin, ofthis invention. In this context, the term “hybridization” refers toconventional hybridization conditions, most preferably stringenthybridization conditions.

The term “functional parts of the BTS02618A toxin” as used herein meansany part(s) or domain(s) of the toxin with a specific structure that canbe transferred to another (Bt) protein for providing a new hybridprotein with at least one functional characteristic (e.g., the bindingand/or toxicity characteristics) of the BTS02618A toxin (Ge et al.,1991). Such parts can form an essential feature of the hybrid Bt proteinwith the binding and/or toxicity characteristics of the BTS02618Aprotein. Such a hybrid protein can have an enlarged host range, animproved toxicity and/or can be used in a strategy to prevent insectresistance development (European Patent Publication (“EP”) 408 403;Visser et al., 1993).

Alternatively, the 5 to 10 Kb fragments, prepared from total DNA of theBTS02617A or BTS02618A or BTS02654B or BTS02652E strain, can be ligatedin suitable expression vectors and transformed in E. coli, and theclones can then be screened by conventional colony immunoprobing methods(French et al., 1986) for expression of the toxin with monoclonal orpolyclonal antibodies raised against the BTS02618A toxin.

Also, the 5 to 10 Kb fragments, prepared from total DNA of the BTS02617Aor BTS02618A or BTS02654B or BTS02652E strain, can be ligated insuitable Bt shuttle vectors (Lereclus et al., 1992) and transformed in acrystal minus Bt-mutant. The clones are then screened for production ofcrystals (detected by microscopy) or crystal proteins (detected bySDS-PAGE).

The so-identified bTS02618A gene was sequenced in a conventional manner(Maxam and Gilbert, 1980) to obtain the DNA sequence. Hybridization inSouthern blots and sequence comparison indicated that this gene isdifferent from previously described genes encoding protoxins and toxinswith activity against Lepidoptera (Höfte and Whiteley, 1989).

An insecticidally effective part of the bTS02618A gene, encoding aninsecticidally effective portion of its protoxin, and a truncated partof the gene, encoding just its toxin, can be made in a conventionalmanner after sequence analysis of the gene. The amino acid sequence ofthe BTS02618A protoxin and toxin was determined from the DNA sequence ofthe bTS02618A gene and the truncated bTS02618A gene. By “aninsecticidally effective part” or “a part” of the bTS02618A gene ismeant a DNA sequence encoding a polypeptide which has fewer amino acidsthan the BTS02618A protoxin but which is still toxic to Lepidoptera.

In order to express all or an insecticidally effective part of thebTS02618A gene or an equivalent gene in E. coli, in other Bt strains andin plants, suitable restriction sites can be introduced, flanking eachgene or gene part. This can be done by site-directed mutagenesis, usingwell-known procedures (Stanssens et al., 1989; White et al., 1989). Inorder to obtain improved expression in plants, it may be preferred tomodify the codon usage of the bTS02618A gene or insecticidally effectivebTS02618A gene part to form an equivalent, modified or artificial geneor gene part in accordance with PCT publications WO 91/16432 and WO93/09218; EP 0,358,962 and EP 0,359,472. For obtaining enhancedexpression in monocot plants such as corn, a monocot intron also can beadded to the bTS02618A chimeric gene, and the DNA sequence of thebTS02618A gene part can be further changed in a translationally neutralmanner, to modify possibly inhibiting DNA sequences present in the genepart by means of site-directed intron insertion and/or by introducingchanges to the codon usage, e.g., adapting the codon usage to that mostpreferred by the specific plant (Murray et al., 1989) without changingsignificantly the encoded amino acid sequence.

The insecticidally effective bTS02618A gene part or its equivalent,preferably the bTS02618A chimeric gene, encoding an insecticidallyeffective portion of the BTS02618A protoxin, can be stably inserted in aconventional manner into the nuclear genome of a single plant cell, andthe so-transformed plant cell can be used in a conventional manner toproduce a transformed plant that is insect-resistant. In this regard, adisarmed Ti-plasmid, containing the insecticidally effective bTS02618Agene part, in Agrobacterium tumefaciens can be used to transform theplant cell, and thereafter, a transformed plant can be regenerated fromthe transformed plant cell using the procedures described, for example,in EP 0,116,718, EP 0,270,822, PCT publication WO 84/02,913 and EuropeanPatent Application (“EPA”) 87/400,544.0 (which are also incorporatedherein by reference), and in Gould et al. (1991). Preferred Ti-plasmidvectors each contain the insecticidally effective bTS02618A gene partbetween the border sequences, or at least located to the left of theright border sequence, of the T-DNA of the Ti-plasmid. Of course, othertypes of vectors can be used to transform the plant cell, usingprocedures such as direct gene transfer (as described, for example in EP0,233,247), pollen mediated transformation (as described, for example inEP 0,270,356, PCT publication WO 85/01856, and U.S. Pat. No. 4,684,611),plant RNA virus-mediated transformation (as described, for example in EP0,067,553 and U.S. Pat. No. 4,407,956), liposome-mediated transformation(as described, for example in U.S. Pat. No. 4,536,475), and othermethods such as the recently described methods for transforming certainlines of corn (Fromm et al., 1990; Gordon-Kamm et al., 1990) and rice(Shimamoto et al., 1989; Datta et al., 1990) and the recently describedmethod for transforming monocots generally (PCT publication WO92/09696).

The resulting transformed plant can be used in a conventional plantbreeding scheme to produce more transformed plants with the samecharacteristics or to introduce the insecticidally effective bTS02618Agene part in other varieties of the same or related plant species.Seeds, which are obtained from the transformed plants, contain theinsecticidally effective bTS02618A gene part as a stable genomic insert.Cells of the transformed plant can be cultured in a conventional mannerto produce the insecticidally effective portion of the BTS02618Aprotoxin, preferably the BTS02618A toxin, which can be recovered for usein conventional insecticide compositions against Lepidoptera (U.S.patent application Ser. No. 821,582; EPA 86/300291.1.).

The insecticidally effective bTS02618A gene part, preferably thetruncated bTS02618A gene, is inserted in a plant cell genome so that theinserted gene is downstream (i.e., 3′) of, and under the control of, apromoter which can direct the expression of the gene part in the plantcell. This is preferably accomplished by inserting the bTS02618Achimeric gene in the plant cell genome. Preferred promoters include: thestrong constitutive 35S promoters (the “35S promoters”) of thecauliflower mosaic virus of isolates CM 1841 (Gardner et al., 1981),CabbB-S (Franck et al., 1980) and CabbB-JI (Hull and Howell, 1987); andthe TR1′ promoter and the TR2′ promoter (the “TR1′. promoter” and “TR2′promoter”, respectively) which drive the expression of the 1′ and 2′genes, respectively, of the T-DNA (Velten et al., 1984). Alternatively,a promoter can be utilized which is not constitutive but rather isspecific for one or more tissues or organs of the plant (e.g., leavesand/or roots) whereby the inserted bTS02618A gene part is expressed onlyin cells of the specific tissue(s) or organ(s). For example, theinsecticidally effective bTS02618A gene part could be selectivelyexpressed in the leaves of a plant (e.g., corn, cotton) by placing theinsecticidally effective gene part under the control of alight-inducible promoter such as the promoter of theribulose-1,5-bisphosphate carboxylase small subunit gene of the plantitself or of another plant such as pea as disclosed in U.S. patentapplication Ser. No. 821,582 and EPA 86/300,291.1. Another alternativeis to use a promoter whose expression is inducible (e.g., by temperatureor chemical factors).

The insecticidally effective bTS02618A gene part is inserted in theplant genome so that the inserted gene part is upstream (i.e., 5′) ofsuitable 3′ end transcription regulation signals (i.e., transcriptformation and polyadenylation signals). This is preferably accomplishedby inserting the bTS02618A chimeric gene in the plant cell genome.Preferred polyadenylation and transcript formation signals include thoseof the octopine synthase gene (Gielen et al., 1984) and the T-DNA gene 7(Velten and Schell, 1985), which act as 3′-untranslated DNA sequences intransformed plant cells.

The insecticidally effective bTS02618A gene part can optionally beinserted in the plant genome as a hybrid gene (EPA 86/300,291.1; Vaecket al., 1987) under the control of the same promoter as a selectablemarker gene, such as the neo gene (EP 0,242,236) encoding kanamycinresistance, so that the plant expresses a fusion protein.

All or part of the bTS02618A gene, encoding an anti-lepidopteranprotein, can also be used to transform other bacteria, such as a B.thuringiensis which has insecticidal activity against Lepidoptera orColeoptera. Thereby, a transformed Bt strain can be produced which isuseful for combatting a wide spectrum of lepidopteran and coleopteraninsect pests or for combatting additional lepidopteran insect pests.Transformation of bacteria with all or part of the bTS02618A gene,incorporated in a suitable cloning vehicle, can be carried out in aconventional manner, preferably using conventional electroporationtechniques as described in Mahillon et al. (1989) and in PCT Patentpublication WO 90/06999.

The BTS02617A, BTS02618A, BTS02654B or BTS02652E strain also can betransformed with all or an insecticidally effective part of one or moreforeign Bt genes such as: the bt18 gene (EP 0,358,557) or another Btgene coding for an anti-Lepidoptera protein; and the bt109P gene (PCTpublication WO 91/16433), coding for an anti-Coleoptera protein.Thereby, a transformed Bt strain can be produced which is useful forcombatting an even greater variety of insect pests (e.g., Coleopteraand/or additional Lepidoptera).

Transformation of the BTS02617A, BTS02618A, BTS02654B or BTS02652Estrain with all or part of a foreign Bt gene, incorporated in aconventional cloning vector, can be carried out in a well known manner,preferably using conventional electroporation techniques (Chassy et al.,1988) or other methods, e.g., as described by Lereclus et al. (1992).

Each of the BTS02617A, BTS02618A, BTS02654B or BTS02652E strains can befermented by conventional methods (Dulmage, 1981; Bernhard and Utz,1993) to provide high yields of cells. Under appropriate conditionswhich are well understood (Dulmage, 1981), the BTS02617A, BTS02618A,BTS02654B and BTS02652E strains each sporulate to produce crystalproteins containing the BTS02168A protoxin in high yields.

An insecticidal, particularly anti-lepidopteran, composition of thisinvention can be formulated in a conventional manner using theBTS02617A, BTS02618A, BTS02654B or BTS02652E strain or preferably theirrespective crystals, crystal proteins or the BTS02168A protoxin, toxinor insecticidally effective protoxin portion as an active ingredient,together with suitable carriers, diluents, emulsifiers and/ordispersants (e.g., as described by Bernhard and Utz, 1993). Thisinsecticide composition can be formulated as a wettable powder, pellets,granules or dust or as a liquid formulation with aqueous or non-aqueoussolvents as a foam, gel, suspension, concentrate, etc. The concentrationof the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, crystals,crystal proteins, or the BTS02618A protoxin, toxin or insecticidallyeffective protoxin portions in such a composition will depend upon thenature of the formulation and its intended mode of use. Generally, aninsecticide composition of this invention can be used to protect a fieldfor 2 to 4 weeks against Lepidoptera with each application of thecomposition. For more extended protection (e.g., for a whole growingseason), additional amounts of the composition should be appliedperiodically.

A method for controlling insects, particularly Lepidoptera, inaccordance with this invention preferably comprises applying (e.g.,spraying), to a locus (area) to be protected, an insecticidal amount ofthe BTS02617A, BTS02618A, BTS02654B or BTS02652E strain, spores,crystals, crystal proteins or the BTS02168A protoxin, toxin orinsecticidally effective protoxin portions, preferably the BTS2168Atoxin. The locus to be protected can include, for example, the habitatof the insect pests or growing vegetation or an area where vegetation isto be grown.

To obtain the BTS02618A protoxin or toxin, cells of the BTS02617A,BTS02618A, BTS02654B or BTS02652E strain can be grown in a conventionalmanner on a suitable culture medium and then lysed using conventionalmeans such as enzymatic degradation or detergents or the like. Theprotoxin can then be separated and purified by standard techniques suchas chromatography, extraction, electrophoresis, or the like. The toxincan then be obtained by trypsin digestion of the protoxin.

The BTS02617A, BTS02618A, BTS02654B or BTS02652E cells can also beharvested and then applied intact, either alive or dead, preferablydried, to the locus to be protected. In this regard, it is preferredthat a purified BTS02617A, BTS02618A, BTS02654B or BTS02652E strain(either alive or dead) be used, particularly a cell mass that is 90.0 to99.9% of the BTS02617A, BTS02618A, BTS02654B or BTS02652E strain.

The BTS02617A, BTS02618A, BTS02654B, or BTS02652E cells, crystals orcrystal proteins or the BTS02618 protoxin, toxin, or insecticidallyeffective protoxin portion can be formulated in an insecticidalcomposition in a variety of ways, using any number of conventionaladditives, wet or dry, depending upon the particular use. Additives caninclude wetting agents, detergents, stabilizers, adhering agents,spreading agents and extenders. Examples of such a composition includepastes, dusting powders, wettable powders, granules, baits and aerosolsprays. Other Bt cells, crystals, crystal proteins, protoxins, toxins,and insecticidally effective protoxin portions and other insecticides,as well as fungicides, biocides, herbicides and fertilizers, can beemployed along with the BTS02617A, BTS02618A, BTS02654B or BTS02652Ecells, crystals or crystal proteins or the BTS02618 protoxin, toxin orinsecticidally effective protoxin portions to provide additionaladvantages or benefits. Such an insecticidal composition can be preparedin a conventional manner, and the amount of the BTS02617A, BTS02618A,BTS02654B or BTS02652E cells, crystals or crystal proteins or theBTS02618A protoxin, toxin or insecticidally effective protoxin portionemployed depends upon a variety of factors, such as the insect pesttargeted, the composition used, the type of area to which thecomposition is to be applied, and the prevailing weather conditions.Generally, the concentration of the BTS02618A protoxin, insecticidallyeffective protoxin portions or toxin will be at least about 0.1% byweight of the formulation to about 100% by weight of the formulation,more often from about 0.15% to about 0.8% by weight of the formulation.

In practice, some insects can be fed the BTS02618A protoxin, toxin,insecticidally effective protoxin portion or mixtures thereof in theprotected area, that is in the area where such protoxin, toxin and/orinsecticidally effective protoxin portion has been applied.Alternatively, some insects can be fed intact and alive cells of theBTS02617A, BTS02618A, BTS02654B or BTS02652E strain or transformantsthereof, so that the insects ingest some of the strain's protoxin andsuffer death or damage.

The following Examples illustrate the invention. The FIGURE and thesequence listing referred to in the Examples are as follows:

The sole FIGURE shows

Southern blot analysis of AluI-digested total DNA of Bt strain HD127(lane 1), the BTS02618A strain (lane 2), Bt strain BTS02459 (containingcrvIA(c),81k, crvIC en cryIE, lane 3), and Bt strain BTS02480E(containing the same genes as HD-127, lane 4), using a mixture ofDNA-probes for cryI crystal protein genes, including the cryIG probe(SEQ ID no. 1). Each band corresponds to a particular crystal proteingene. With these probes, the BTS02618A strain is found to contain thecrvIA(b) gene and a novel gene, which is the bTS02618A gene, identifiedby an AluI fragment of approximately 530 bp, hybridizing to the cryIGprobe of SEQ ID no. 1. The names of the recognized cryI genes areindicated, as well as the size of some fragments. The bTS02618A gene isindicated with three asterisks; “?” indicates an unknown gene fragment.

SEQUENCE LISTING

SEQ ID No. 1—Nucleotide sequence of the DNA probe used to isolate thebTS02618A gene. This probe is derived from part of the cryIG DNAsequence and is complementary to nucleotides 2732-2750 of the DNAsequence described by Smulevitch et al. (1991).

SEQ ID No. 2—The 5′ partial nucleotide sequence of the bTS02618A gene,comprising the presumptive translation initiation codon at nucleotideposition 195-197.

SEQ ID No. 3—The 3′ partial nucleotide sequence of the bTS02618A gene(N: unknown nucleotide), comprising the presumptive translational stopcodon at nucleotide position 1146-1148.

SEQ ID No. 4—The nucleotide sequence of the bTS02618A gene and thetranslated amino acid sequence of the BTS02618A protoxin. The openreading frame of the protoxin reaches from nucleotide 668 to nucleotide4141. The translation initiation codon is at nucleotide position668-670, the translation stop codon is at nucleotide position 4139-4141.

Unless otherwise stated in the Examples, all procedures for making andmanipulating recombinant DNA are carried out by the standardizedprocedures described in Sambrook et al., Molecular Cloning—A LaboratoryManual. Second Ed., Cold Spring Harbor Laboratory Press, New York(1989).

By stringent hybridization conditions is meant that the filters areprehybridized for 1 to 2 hours in either 50% formamide, 5×SSPE,2×Denhardt's reagent and 0.1% SDS at 42° C. or 6×SSX, 2×Denhardt'sreagent and 0.1% SDS at 68° C. The radiolabeled probe is then addeddirectly to the prehybridization fluid and incubation is carried out for16 to 24 hours at the appropriate temperature. After incubation, thefilters are then washed for 20 minutes at room temperature in 1×SSC,0.1% SDS, followed by three washes of 20 minutes each at 68° C. in0.2×SSC and 0.1% SDS. An autoradiograph is established by exposing thefilters for 24 to 48 hours to X-ray film (Kodak XAR-2 or equivalent) at−70° C. with an intensifying screen.

EXAMPLE 1 Characterization of the BTS02617A, BTS02618A, BTS02654B andBTS02652E Strains

The BTS02617A, the BTS02618A and the BTS02654B strain were isolated fromgrain dust sampled in Cadlan, province of Bicol, The Philippines andwere deposited at the BCCM-LMG on Jul. 2, 1992 under accession Nos. LMGP-12592, LMG P-12593 and LMG P-12594, respectively. Strain BTS02652E wasalso isolated from Philippine grain dust, and was deposited at theBCCM-LMG on Mar., 1, 1993 under accession No. LMG P-13493.

Each strain can be cultivated on conventional standard media, preferablyT₃ medium (tryptone 3 g/l, tryptose 2 g/l, yeast extract 1.5 g/l, 5 mgMnCl₂, 0.05 M Na₂PO₄, pH 6.8 and 1.5% agar), preferably at 28° C. Forlong term storage, it is preferred to mix an equal volume of aspore-crystal suspension with an equal volume of 50% glycerol and storethis at −70° C. or lyophilize a spore-crystal suspension. Forsporulation, growth on T₃ medium is preferred for 48 hours at 28° C.,followed by storage at 4° C. During its vegetative phase, each of thestrains can also grow under facultative anaerobic conditions, butsporulation only occurs under aerobic conditions.

Sterilization of each strain occurs by autoclave treatment at 120° C. (1bar pressure) for 20 minutes. Such treatment totally inactivates thespores and the BTS02617A,BTS02618A, BTS02654B, and BTS02652E protoxins.UV radiation (254 nm) also inactivates the spores.

After cultivating on Nutrient Agar (“NA”, Difco Laboratories, Detroit,Mich., USA) for one day, colonies of each of the BTS02617A, BTS02618A,BTS02654B and BTS02652E strains form opaque white colonies withirregular edges. Cells of each strain (Gram positive rods of1.7-2.4×5.6-7.7 μm) sporulate after 48 hrs cultivation at 28° C. on T₃agar. The crystal proteins produced during sporulation are packaged incrystals of the BTS02617A, BTS02618A, BTS02654B, and BTS02652E strains.Quite remarkably, the crystal remains attached to the spore aftersporulation.

The Bt serotype of the BTS02617A, BTS02618A, BTS02645B and BTS02652Estrains was determined to be serotype tolworthi H9 of all these strainswhich was determined by conventional serotyping methods as conducted bythe WHO Collaborating Center for Entomopathogenic Bacillus.

EXAMPLE 2 Insecticidal Activity of the BTS02617A, BTS02618A, BTS02654Band BTS02652E Strains and the BTS02618A Protoxin Against Noctuidae spp.,Yponomeutidae spp. and Pyralidae spp.

Toxicity assays were performed on neonate larvae (for Plutellaxylostella, third instar larvae were used) fed on an artificial dietlayered with spore-crystal mixtures from one of the BTS02617A,BTS02618A, BTS02654B and BTS02652E strains or the BTS02618A protoxin ortoxin. The artificial diet was dispensed in wells of Costar 24-wellplates. Formaldehyde was omitted from the diet. 50 μl of a sampledilution was applied on the surface of the diet and dried in a laminarair flow. For LC₅₀ assays, the dilutions were made in a PBS-BSA buffer,and five dilutions were applied. Two larvae were placed in each well and24 larvae were used per sample dilution. Dead and living M. brassica, S.frugiperda, H. virescens, O. nubilalis, Plutella xylostella and S.exigua larvae were counted on the fifth day, and dead and living A.ipsilon and S. littoralis larvae were counted on the sixth day. The LC₅₀and LC₉₅ values (the concentrations required to kill respectively 50% or95% of the insects tested, expressed in number of spore-crystals/cm² orng (pro)toxin/cm²) were calculated using Probit-analysis (Finney, 1971),and the results are set forth below.

Spodoptera littoralis

Experiment/Strain LC₅₀ ^(a) LC₉₅ ^(a) FL_(min-max) ^(b) Slope Experiment1 BTS02618A 2.4 7.7 1.5-3.4 3.2 HD127^(c) 2.5 168 1.2-7.4 1.0 Experiment2 BTS02618A 1.1 4 0.8-1.6 3.0 HD127 21.2 133.7 14.4-31.9 2.0 ^(a)10⁵spore-crystals per cm² ^(b)95% fiducial limits of LC₅₀ values ^(c)fromthe Howard Dulmage collection, housed at the Northern Region ResearchCenter, 1815 North University, Peoria, Ill, USA. The curator is Dr. L.Nakamura.

Experiments with purified BTS02618A protoxin also show a significanttoxicity of this protoxin against S. littoralis larvae.

Spodoptera exigua

1. Crystal/Spore Mixtures

Experiment/Strain LC₅₀ ^(a) LC₉₅ ^(a) FL_(min-max) ^(b) Slope Experiment1 BTS02618A 1.4 7.9 0.48-3.9  2.2 HD127 8.2 163.5  5.1-15.7 1.3Experiment 2 BTS02618A 1.2 3.56 0.91-1.57 3.5 BTS02617A 0.79 2.120.61-1.03 3.81 HD127 3.5 44.2  1.36-11.5* 1.5 Florbac 4.1 53.9 1.5-17.0* 1.47 BTS00170U^(c) 5.1 46.5  1.83-24.4* 1.71 Experiment 3Javelin^(d) 23.12 195.7 14.6-56.7 1.77 Experiment 4 BTS02618A 1.07 2.910.83-1.39 3.8 BTS02617A 0.87 4.7 0.59-1.21 2.22 HD127 4.7 56.9 1.85-18.7* 1.52 Florbac^(e) 2.53 48.1  0.79-6.71* 1.29 BTS00170U 1.9456.3 0.55-5.4* 1.12 ^(a)10⁵ spore-crystals per cm² ^(b)95% fiduciallimits of LC₅₀ values, values marked with * are 90% fiducial limits ofLC₅₀ values ^(c)PCT patent publication WO 90/06999 ^(d)strain isolatedfrom Javelin ® (Sandoz, Lichtstrasse, Basel, Switzerland) ^(e)strainfrom Florbac ® (Novo Nordisk, Novo All{dot over (e)}, Bagsvaerd,Denmark)

2. Toxin/Protoxin Assays.

ICP LC₅₀ ^(a) LC₉₅ ^(a) FL_(min-max) ^(b) Slope BTS02618A Protoxin 26.6100.6 20.9-33.9 2.8 CryIC Toxin 68.9 313.2 50.5-94.1 2.5 CryID Toxin118.6 870.6  82.7-170.0 1.9 ^(a)ng/cm² ^(b)95% fiducial limits of LC₅₀values

Mamestra brassica

1. Crystal/Spore Mixtures.

Experiment/Strain LC₅₀ ^(a) LC₉₅ ^(a) FL_(min-max) ^(b) Slope HD127 37.8297.6 17.8-91.1 1.8 BTS02618A 8.6 59.6  6.0-12.2 1.9 BTS02617A 5.2 25.83.7-7.1 2.4 BTS02652E 12.9 44.2  9.7-17.2 3.0 BTS02654B 14.2 60.510.8-19.9 2.6 ^(a)10⁵ spore-crystals per cm² ^(b)95% fiducial limits ofLC₅₀ values

2. Protoxin Assays.

ICP LC₅₀ ^(a) LC₉₅ ^(a) FL_(min-max) ^(b) Slope BTS02618A Protoxin 25.3125.1 19.3-33.2 2.4 CryIC Protoxin 22.0 62.9 16.3-29.6 3.6 CryIA (b)Protoxin 162.4 7169  93.2-283.1 1.0 ^(a)ng/cm² ^(b)95% fiducial limitsof LC₅₀ values

Agrotis ipsilon

1. Crystal/Spore Mixtures.

Strain mortality^(a) genes^(b) Btgall.^(c)  1/20 cryIF, cryIG, cryII,81k HD127^(d)  2/20 cryIAa, cryIAb, cryIC, cryID, cryII, 81k BTS02618A16/20^(e) cryIAb, cryII, bTS02618A Buffer  1/20 none ^(a)number of 1stinstar larvae killed after 6 days (10⁷ spore-crystals per cm²) ^(b)genesknown to be present in these strains ^(c)Btgall. as described bySmulevitch et al (1991) ^(d)HD127 is available at the Howard DulmageCollection (NRRC, see above) ^(e)surviving larvae show severegrowth-inhibition

STRAIN LC₅₀ ^(a) LC₉₅ ^(a) FL_(min-max) ^(b) Slope BTS02618A 84.4 207.965.9-109.6 4.2 HD127 >250 BTS02617A 53.4 261.0 27.7-112.3 2.4 ^(a)10⁶spores/cm² ^(b)95% fiducial limits of LC₅₀ values

2. Toxin/Protoxin Assay.

ICP LC₅₀ ^(a) LC₉₅ ^(a) FL_(min-max) ^(b) Slope CryIAc Toxin >1350BTS02618A Protoxin 212.2 1973 168.1-267.9 1.7 ^(a)ng/cm² ^(b)95%fiducial limits of LC₅₀ values

Since MacIntosh et al. (1990) described some activity of the CryIActoxin towards A. ipsilon, purified CryIAc toxin was tested on thisinsect for comparison but did not cause any significant mortality of A.ipsilon.

Heliothis virescens

1. Crystal/Spore Mixture.

Experiment/Strain LC₅₀ ^(a) LC₉₅ ^(a) FL_(min-max) ^(b) Slope BTS02617A1.69 14.99 0.67-2.89 1.73 BTS02618A 2.71 25.4 0.88-6.99 1.69BTS00170U^(c) 15.1 398.7  8.3-41.2 1.15 Dipel^(d) 2.99 14.11 1.25-7.762.45 ^(a)10³ spore-crystals per cm² ^(b)95% fiducial limits of LC₅₀values ^(c)PCT patent publication WO 90/06999 ^(d)strain isolated fromDipel ™ (Abbott Laboratories, North Chicago, Ill., USA)

2. Toxin/Protoxin Assay.

ICP LC₅₀ ^(a) FL_(min-max) ^(b) LC₉₅ ^(a) Slope BTS02618A Protoxin 31.620-50 182.7 2.1 CryIAb Toxin 7.2  4.9-10.5 169.1 1.2 ^(a)ng/cm² ^(b)95%fiducial limits of LC₅₀ values

Ostrinia nubilalis

1. Crystal/Spore Mixtures.

Experiment/Strain LC₅₀ ^(a) LC₉₅ ^(a) FL_(min-max) ^(b) Slope BTS02617A4.92 12.49 2.45-6.81 4.0 BTS02618A 6.17 39.7 2.93-9.74 2.0 Dipel^(c) >30^(a)10⁵ spore-crystals per cm² ^(b)95% fiducial limits of LC₅₀ values^(c)strain isolated from Dipel ™ (Abbott Laboratories)

2. Purified Protoxin Assay

ICP 100% Mortality^(a) CryIAb Toxin 1350 CryIB Toxin 1350 BTS02618AProtoxin 100 ^(a)concentration at which 100% mortality was observed (inng/cm²)

The purified BTS02618A protoxin also showed a significant toxicity toOstrinia nubilalis larvae, as compared with the CryI toxins that aremost active against Ostrinia.

Plutella xylostella

Plutella xylostella larvae also showed significant mortality afterapplication of purified BTS02618A toxin to their artificial diet inseveral experiments.

Spodoptera frugiperda

Crystal/Spore Mixtures of a bTS02618A gene-transformed crystal-minus Btstrain (Mahillon et al., 1989) were also found to significantly inhibitlarval growth of S. frugiperda larvae in insect feeding trials.

In conclusion, the strains of this invention and the BTS02618A proteinof this invention have a strong insecticidal activity against a broadrange of insects that are not susceptible to any single currentlyavailable Bt protein and have an activity against at least threeSpodoptera spp. and against other Noctuidae, such as A. ipsilon, M.brassica and H. virescens, as well as against Pyralidae, such as O.nubilalis and Yponomeutidae such as Plutella xylostella. These resultsare summarized and compared with results for other CryI genes (VanFrankenhuyzen, 1993) in Table 1 which shows the unique range of insectssusceptible to the BTS02618A protein.

EXAMPLE 3 Identification of the bTS02618A Gene

The bTS02618A gene was identified in the BTS02618A strain by Southernblot analysis (FIG. 1) of AluI digested total DNA of the strain using,as a DNA probe, the DNA sequence of the cryIG gene (Gleave et al., 1992)of SEQ ID No. 1 and using standard hybridization conditions. Partial DNAsequences of the bTS02618A gene, showing its 5′ and 3′ end portions, areshown in SEQ ID Nos. 2 and 3, respectively, and the full DNA sequence ofthe bTS02618A gene and the full amino acid sequence of the BTS02618Aprotein are shown in SEQ ID No. 4.

The partial sequences of SEQ ID Nos. 2 and 3 allow the bTS02618A gene tobe recognized in the BTS02617A, BTS02654B and BTS02652E strains andallow the construction of probes to identify and isolate the full genesequence in these and other Bt strains. The translation initiation codonof the bTS02618A gene is identified at nucleotide position 195-197 inSEQ ID No. 2, corresponding to nucleotide position 668-670 in SEQ IDNo.4. The translation stop codon is identified at nucleotide position1146-1148 in SEQ ID No. 3, corresponding to nucleotide position4139-4141 in SEQ ID No. 4.

The bTS02618A gene was also identified in the BTS02617A, BTS02654B andBTS02652E strains by using the DNA sequence of SEQ ID No. 1 as a probe,as well as other DNA probes of conserved DNA fragments in cryI genes.

The full length bTS02618A gene was found to encode a 129.9 kD protoxin.A comparison of the amino acid sequence with other known CryI proteinsshowed that the C-terminal part (C-terminal of conserved sequence block5) was homologous with CryIG (88%). The best homology for the N-terminalpart (the toxin) was found with the CryIB toxin, but this was found tobe less than 50% (homology is expressed as the number of perfect matchesdivided by the number of amino acids of the longest fragment).

The smallest insecticidal protein is believed to be a 69 kD (615 aminoacids) protein stretching from amino acid number 44 to amino acid number658 in SEQ ID No. 4. A smaller tryptic fragment of 55 kD (494 aminoacids), stretching from amino acid number 165 to amino acid number 658in SEQ ID No. 4, still has insecticidal activity towards S. exigua, butthis activity is significantly reduced. Thus, a truncated bTS02618A geneor an equivalent truncated gene preferably encodes the 69 kD protein ofthe BTS02618A protoxin of SEQ ID No.4 as described above.

EXAMPLE 4 Cloning and Expression of the bTS02618A Gene

In order to isolate the bTS02618A gene, total DNA from the BTS02618Astrain was prepared and partially digested with Sau3A. The digested DNAwas size fractionated on a sucrose gradient and fragments ranging from 7Kb to 10 Kb were ligated to the BamH1-digested and BAP-treated cloningvector pUC19 (Yannisch-Perron et al., 1985). Recombinant E.coli clonescontaining the vector were then screened with the cryIG DNA probe of SEQID No. 1 which is described in Example 3, to identify clones containingthe bTS02618A gene.

The so-identified DNA fragments were then sequenced according to Maxamand Gilbert (1980). Partial sequences of the bTS02618A gene are shown inSEQ ID Nos. 2 and 3, and a full sequence of the bTS02618A gene and theBTS02618A protein is shown in SEQ ID No. 4. Based on the DNA sequenceanalysis, the gene is cut with appropriate restriction enzymes to givethe truncated bTS02618A gene encoding the BTS02618A toxin. Expression ofthe gene in E.coli was induced using standard procedures (Sambrook etal., 1989, supra).

The bTS02618A gene is also introduced by routine procedures into acrystal-minus Bt strain, using Bt plasmids PGI2 or PGI3 (Mahillon andSeurinck 1988; Mahillon et al., 1988).

EXAMPLE 5 Insertion of the bTS02618A Gene and the Truncated bTS02618AGene in E. coli and Insertion of the Truncated bTS02618A Gene in Plants.

In order to express the bTS02618A gene and the truncated bTS02618A geneof Example 4 in E. coli and in plants, different gene cassettes are madein E. coli according to the procedure described in EPA 86/300291.1 andEPA 88/402115.5.

To allow significant expression in plants, cassettes containing a) thetruncated gene or b) a hybrid gene that is a fusion of i) the truncatedgene and ii) the neo gene are each: inserted between the T-DNA bordersequences of intermediate plant expression vectors as described in EPA86/300291.1; fused to transcript formation and polyadenylation signalsin the plant expression vectors; placed under the control of theconstitutive promoter from cauliflower mosaic virus driving the 35S3transcript (Hull and Howell, 1987) or the 2′ promoter from the TR-DNA ofthe octopine Ti-plasmid (Velten et al., 1984); and fused to 3′ endtranscript formation and polyadenylation signals of the octopinesynthase gene (Gielen et al., 1984).

Using standard procedures (Deblaere et al., 1985), the intermediateplant expression vectors, containing the truncated bTS02618A gene, aretransferred into the Agrobacterium strain C58C1Rif^(R) (U.S. patentapplication Ser. No. 821,582; EPA 86/300,291.1) carrying the disarmedTi-plasmid pGV2260 (Vaeck et al., 1987). Selection for spectinomycinresistance yields cointegrated plasmids, consisting of pGV2260 and therespective intermediate plant expression vectors. Each of theserecombinant Agrobacterium strains is then used to transform differentcotton plants so that the truncated bTS02618A gene is contained in, andexpressed by, different plant cells.

EXAMPLE 6 Expression of the Truncated bTS02618A Gene in Slants

The insecticidal activity against Lepidoptera of the expression productsof the truncated bTS02618A gene in leaves of transformed plants,generated from the transformed plant cells of Example 5, is evaluated byrecording the growth rate and mortality of Agrotis and Spodoptera spp.larvae fed on these leaves. These results are compared with the growthrate of larvae fed leaves from untransformed plants. Toxicity assaysagainst Agrotis and Spodoptera spp. are performed as described in EP0,358,557, U.S. patent application Ser. No. 821,582 and EPA86/300,291.1. A significantly higher mortality rate is obtained amonglarvae fed on leaves of transformed plants containing the truncatedbTS02618A gene and the truncated bTS02618A-neo hybrid gene than amonglarvae fed the leaves of untransformed plants. The transformed plantsare also found to resist Ostrinia nubilalis Mamestra brassica, Heliothisvirescens and Plutella xylostella attack by their expression of theBTS02618A protein.

Needless to say, this invention is not limited to the BTS02617A strain(BCCM-LMG P-12592), the BTS02618A strain (BCCM-LMG P-12593), theBTS02654B strain (BCCM-LMG P-12594) and the BTS02652E (BCCM-LMG P-13493)strain. Rather, the invention also includes any mutant or variant of theBTS02617A, BTS02618A, BTS02654B, and BTS02652E strain which producescrystals, crystal proteins, protoxin or toxin having substantially thesame properties, particularly anti-Lepidoptera properties, quiteparticularly anti-Noctuidae, anti-Yponomeutidae and anti-Pyralidaeproperties, especially anti-Spodoptera, anti-Plutella, anti-Ostriniaanti-Mamestra; anti-Heliothis and anti-Agrotis properties, as therespective BTS02617A, BTS02618A, BTS02654B or BTS02652E crystals orcrystal proteins, or the BTS02618A protoxin or toxin. This inventionalso includes the bTS02618A gene and any insecticidally effective partsthereof, like the truncated bTS02618A gene. In this regard, the term“bTS02618A gene” as used herein means the gene isolated from theBTS02617A, BTS02618A, BTS02654B or BTS02652E strain and hybridizing tothe nucleotide sequence of SEQ ID No. 1 and any equivalent gene encodinga protoxin having substantially the same amino acid sequence andinsecticidal activity as the BTS02618A protoxin and preferablycontaining the partial nucleotide sequences shown in SEQ ID Nos. 2 and3, or the full sequence shown in SEQ ID No. 4.

This invention also is not limited to cotton plants transformed with thetruncated bTS02618A gene. It includes any plant, such as tomato,tobacco, rapeseed, alfalfa, sunflower, lettuce, potato, corn, rice,soybean, Brassica species, sugar beet and other legumes and vegetables,transformed with an insecticidally effective part of the bTS02618A geneor an equivalent gene.

Nor is this invention limited to the use of Agrobacterium tumefaciensTi-plasmids for transforming plant cells with an insecticidallyeffective bTS02618A gene part. Other known techniques for plant celltransformations, such as by means of liposomes, by electroporation or byvector systems based on plant viruses or pollen, can be used fortransforming monocotyledons and dicotyledons with such a gene part.

Furthermore, DNA sequences other than those present naturally in theBTS02617A, BTS02618A, BTS02654B and BTS02652E strains and encoding theBTS02618A protoxin and toxin can be used for transforming plants andbacteria. In this regard, the natural DNA sequence of these genes can bemodified by: 1) replacing some codons with others that code either forthe same or different, preferably the same, amino acids; 2) deleting oradding some codons; and/or 3) reciprocal recombination as described byGe et al. (1991); provided that such modifications do not substantiallyalter the properties, particularly the insecticidal properties,especially anti-lepidoptera properties, of the encoded, insecticidallyeffective portions of the BTS02618A protoxin (e.g., toxin). For example,an artificial bTS02618A gene or gene part of this invention, asdescribed above, having a modified codon usage, could be used in certaincircumstances instead of a natural insecticidally effective bTS02618Agene part in a bTS02618A chimeric gene of this invention fortransforming plants.

Also, other DNA recombinants containing all or part of the bTS02618Agene in association with other foreign DNA, particularly the DNA ofvectors suitable for transforming plants and microorganisms other thanE. coli, are encompassed by this invention. In this regard, thisinvention is not limited to the specific plasmids containing thebTS02618A gene, or parts thereof, that were heretofore described, butrather, this invention encompasses any DNA recombinants containing DNAsequences that are their equivalent. Further, the invention relates toall DNA recombinants that include all or part of the bTS02618A gene andthat are suitable for transforming microorganisms (e.g., plantassociated bacteria such as other Bacillus thuringiensis strains,Bacillus subtilis, Pseudomonas, and Xanthomonas or yeasts such asStreptomyces cerevisiae) under conditions which enable all or part ofthe gene to be expressed and to be recoverable from said microorganismsor to be transferred to a plant cell.

TABLE 1 Activity of CryI proteins towards several lepidopteran insectpests: + and − indicates the presence or absence of insecticidalactivity, +/− indicates low activity (according to Van Frankenhuyzen(1993)), NA indicates no data available, the protein BTS02618A isabbreviated as 2618A (data of Van Frankenhuyzen (1993) and thisinvention (for A. ipsilon and 2618A)). 2618A IAb IAc IB IC IF S.exigua + +/− − − + + S. littoralis + − − − + NA H. virescens + + + −+/− + A. ipsilon + NA − NA NA NA O. nubilalis + + + NA NA + P.xylostella + + + + + NA M. brassica + + − − + NA

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Van Frankenhuyzen,“The Challenge of Bacillus thuringiensis”, in“Bacillus thuringiensis, An Environmental Biopesticide: Theory andPractice”, pp.1-35, eds. Entwistle, P. F., Cory, J. S., Bailey, M. J.and Higgs, S., John Wiley and Sons, New York (1993).

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Yannisch-Perron, C., Vierra, J. and Messing, J., Gene 33, 103-119(1985).

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 5(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single(D) TOPOLOGY: linear (ii) MOLECULE TYPE: other nucleic acid (A)DESCRIPTION: /desc = “synthetic DNA” (ix) FEATURE: (A) NAME/KEY:misc_feature (B) LOCATION:1..19 (D) OTHER INFORMATION:/function= “forisolating bTS02618A gene from its containing strain” /note= “the probeis a part of the coding DNA strand of the cryIG gene (Smulevitch et al.(1991)” (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..19 (D)OTHER INFORMATION:/note= “this probe is used to isolate the bTS02618Agene from its containing strain” (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: TTCTGTACTA TTGATTGTA 19 (2) INFORMATION FOR SEQ ID NO: 2: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 1561 base pairs (B) TYPE: nucleicacid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE:DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINALSOURCE: (A) ORGANISM: Bacillus thuringiensis (B) STRAIN: BTS02618A (ix)FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..1561 (D) OTHERINFORMATION:/note= “contains the translation initiation codon of thebTS02618A gene” (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: AAAAAGAAATAGGAATAAAT ACTATCCATT TTTTCAAGAA ATATTTTTTT ATTAGAAAGG 60 AATCTTTCTTACACGGGAAA ATCCTAAGAT TGAGAGTAAA GATATATATA TATAAATACA 120 ATAAAGAGTTTGTCAGGATT TTTGAAAGAT ATGATATGAA CATGCACTAG ATTTATAGTA 180 TAGGAGGAAAAAGTATGAAT CGAAATAATC AAAATGAATA TGAAATTATT GATGCCCCCC 240 ATTGTGGGTGTCCATCAGAT GACGATGTGA GGTATCCTTT GGCAAGTGAC CCAAATGCAG 300 CGTTACAAAATATGAACTAT AAAGATTACT TACAAATGAC AGATGAGGAC TACACTGATT 360 CTTATATAAATCCTAGTTTA TCTATTAGTG GTAGAGATGC AGTTCAGACT GCGCTTACTG 420 TTGTTGGGAGAATACTCGGG GCTTTAGGTG TTCCGTTTTC TGGACAAATA GTGAGTTTTT 480 ATCAATTCCTTTTAAATACA CTGTGGCCAG TTAATGATAC AGCTATATGG GAAGCTTTCA 540 TGCGACAGGTGGAGGAACTT GTCAATCAAC AAATAACAGA ATTTGCAAGA AATCAGGCAC 600 TTGCAAGATTGCAAGGATTA GGAGACTCTT TTAATGTATA TCAACGTTCC CTTCAAAATT 660 GGTTGGCTGATCGAAATGAT ACACGAAATT TAAGTGTTGT TCGTGCTNAA TTTATAGCTT 720 TAGACCTTGATTTTGTTAAT GCTATTCCAT TGTTTGCAGT AAATGGACAG CAGGTTCCAT 780 TACTGTCAGTATATGCACAA GCTGTGAATT TACATTTGTT ATTATTAAAA GATGCATCTC 840 TTTTTGGAGAAGGATGGGGA TTCACACAGG GGGAAATTTC CACATATTAT GACCGTCAAT 900 TGGAACTAACCGCTAAGTAC ACTAATTACT GTGAAACTTG GTATAATACA GGTTTAGATC 960 GTTTAAGAGGAACAAATACT GAAAGTTGGT TAAGATATCA TCAATTCCGT AGAGAAATGA 1020 CTTTAGTGGTATTAGATGTT GTGGCGCTAT TTCCATATTA TGATGTACGA CTTTATCCAA 1080 CGGGATCAAACCCACAGCTT ACACGTGAGG TATATACAGA TCCGATTGTA TTTAATCCAC 1140 CAGCTAATGTTGGACTTTGC CGACGTTGGG GTACTAATCC CTATAATACT TTTTCTGAGC 1200 TCGAAAATGCCTTCATTCGC CCACCACATC TTTTTGATAG GCTGAATAGC TTAACAATCA 1260 GCAGTAATCGATTTCCAGTT TCATCTAATT TTATGGATTA TTGGTCAGGA CATACGTTAC 1320 GCCGTAGTTATCTGAACGAT TCAGCAGTAC AAGAAGATAG TTATGGCCTA ATTACAACCA 1380 CAAGAGCAACAATTAATCCC GGAGTTGATG GAACAAACCG CATAGAGTCA ACGGCAGTAG 1440 ATTTTCGTTCTGCATTGATA GGTATATATG GCGTGAATAG AGCTTCTTTT GTCCCAGGAG 1500 GCTTGTTTAATGGTACGACT TCTCCTGCTA ATGGAGGATG TAGAGATCTC TATGATACAA 1560 A 1561 (2)INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1554 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D)TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL:NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Bacillusthuringiensis (B) STRAIN: BTS02618A (ix) FEATURE: (A) NAME/KEY:misc_feature (B) LOCATION:1146..1148 (D) OTHER INFORMATION:/note=“Presumed translational stop codon of bTS02618A gene” (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 3: AAAATTATCC AACATACATT TATCAAAAAG TAGATGCATCGGTGTTAAAG CCTTATACAC 60 GCTATAGACT AGATGGATTT GTGAAGAGTA GTCAAGATTTAGAAATTGAT CTCATCCACC 120 ATCATAAAGT CCATCTTGTA AAAAATGTAC CAGATAATTTAGTATCTGAT ACTTACTCAG 180 ATGGTTCTTG CAGCGGAATC AACCGTTGTG ATGAACAGCATCAGGTAGAT ATGCAGCTAG 240 ATGCGGAGCA TCATCCAATG GATTGCTGTG AAGCGGCTCAAACACATGAG TTTTCTTCCT 300 ATATTAATAC AGGGGATCTA AATGCAAGTG TAGATCAGGGCATTTGGGTT GTATTAAAAG 360 TTCGAACAAC AGATGGGTAT GCGACGTTAG GAAATCTTGAATTGGTAGAG GTTGGGCCAT 420 TATCGGGTGA ATCTCTAGAA CGGGAACAAA GAGATAATGCGAAATGGAAT GCAGAGCTAG 480 GAAGAAAACG TGCAGAAATA GATCGTGTGT ATTTAGCTGCGAAACAAGCA ATTAATCATC 540 TGTTTGTAGA CTATCAAGAT CAACAATTAA ATCCAGAAATTGGGCTAGCA GAAATTAATG 600 AAGCTTCAAA TCTTGTAGAG TCAATTTCGG GTGTATATAGTGATACACTA TTACAGATTC 660 CTGGGATTAA CTACGAAATT TACACAGAGT TATCCGATCGCTTACAACAA GCATCGTATC 720 TGTATACGTC TAGAAATGCG GTGCAAAATG GAGACTTTAACAGTGGTCTA GATAGTTGGA 780 ATACAACTAT GGATGCATCG GTTCAGCAAG ATGGCAATATGCATTTCTTA GTTCTTTCGC 840 ATTGGGATGC ACAAGTTTCC CAACAATTGA GAGTAAATCCGAATTGTAAG TATGTCTTAC 900 GTGTGACAGC AAGAAAAGTA GGAGGCGGAG ATGGATACGTCACAATCCGA GATGGCGCTC 960 ATCACCAAGA AACTCTTACA TTTAATGCAT GTGACTACGATGTAAATGGT ACGTATGTCA 1020 ATGACAATTC GTATATAACA GAAGAAGTGG TATTCTACCCAGAGACAAAA CATATGTGGG 1080 TAGAGGTGAG TGAATCCGAA GGTTCATTCT ATATAGACAGTATTGAGTTT ATTGAAACAC 1140 AAGAGTAGAA GAGGGGGATC CTAACGTATA GCAACTATGAGAGGATACTC CGTACAAACA 1200 AAGATTAAAA AAAGGTAAAA TGAATAGAAC CCCCTACTGGTAGAAGGACC GATAGGGGGT 1260 TCTTACATGA AAAAATGTAG CTGTTTACTA AGGTGTATAAAAAACAGCAT ATCTGATAGA 1320 AAAAAGTGAG TACCTTATAA AGAAAGAATT CCATTCACAGTTTCGGTATC ATATAAATAA 1380 TGATAGGGGT ATCCTTCTTA TTTACATTAT TTTTCGCAATTATCTCGACG TTCTTCTTTC 1440 CGCTCACAAT GATGATGATC ATGACAACAA TCGCGTCCATAGCGAACTCT TTCGATATTA 1500 ATAATATCTA AACTCGTGTA GCAGTCATTT CCATTTTTTTTGATCCAGTA AATA 1554 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 4344 base pairs (B) TYPE: nucleic acid (C)STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA(genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION:668..4141 (ix)FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..4344 (D) OTHERINFORMATION:/note= “encompasses entire sequence of SEQ ID NO (SID) 2:from nt position 474 to 2034 in SID 4” (ix) FEATURE: (A) NAME/KEY:misc_feature (B) LOCATION:1..4344 (D) OTHER INFORMATION:/note= “alsoencompasses part of the sequence of SID 3: from nt position 2994 to 4344in SID 4” (ix) FEATURE: (A) NAME/KEY: misc_feature (B) LOCATION:1..4344(D) OTHER INFORMATION:/note= “SID 3 shows additional nucleotides,located 3′ from the sequence shown in SID 4 (1352-1554 in SID 4)” (xi)SEQUENCE DESCRIPTION: SEQ ID NO: 4: GAATTCGAGC TCGGTACCTT TTCAGTGTATCGTTTCCCTT CCATCAGGTT TTCAAATTGA 60 AAAGCCGAAT GATTTGAAAC TTGTTTACGATGTAAGTCAT TTGTCTATGA CGAAAGATAC 120 GTGTAAAAAA CGTATTGAGA TTGATGAATGTGGACAAGTA GAAATTGACT TACAAGTATT 180 AAAGATTAAG GGTGTCCTTT CTTTTATCGGAAATTTCTCT ATTGAACCTA TTCTGTGTGA 240 AAACATGTAT ACAACGGTTG ATAGAGATCCGTCTATTTCC TTAAGTTTCC AAGATACGGT 300 ATATGTGGAC CATATTTTAA AATATAGCGTCCAACAACTA CCATATTATG TAATTGATGG 360 TGATCATATT CAAGTACGTG ATTTACAAATCAAACTGATG AAAGAGAATC CGCAATCTGC 420 TCAAGTATCA GGTTTGTTTT GTTTTGTATATGAGTAAGAA CCGAAGGTTT GTAAAAAAGA 480 AATAGGAATA AATACTATCC ATTTTTTCAAGAAATATTTT TTTATTAGAA AGGAATCTTT 540 CTTACACGGG AAAATCCTAA GATTGAGAGTAAAGATATAT ATATATAAAT ACAATAAAGA 600 GTTTGTCAGG ATTTTTGAAA GATATGATATGAACATGCAC TAGATTTATA GTATAGGAGG 660 AAAAAGT ATG AAT CGA AAT AAT CAA AATGAA TAT GAA ATT ATT GAT GCC 709 Met Asn Arg Asn Asn Gln Asn Glu Tyr GluIle Ile Asp Ala 1 5 10 CCC CAT TGT GGG TGT CCA TCA GAT GAC GAT GTG AGGTAT CCT TTG GCA 757 Pro His Cys Gly Cys Pro Ser Asp Asp Asp Val Arg TyrPro Leu Ala 15 20 25 30 AGT GAC CCA AAT GCA GCG TTA CAA AAT ATG AAC TATAAA GAT TAC TTA 805 Ser Asp Pro Asn Ala Ala Leu Gln Asn Met Asn Tyr LysAsp Tyr Leu 35 40 45 CAA ATG ACA GAT GAG GAC TAC ACT GAT TCT TAT ATA AATCCT AGT TTA 853 Gln Met Thr Asp Glu Asp Tyr Thr Asp Ser Tyr Ile Asn ProSer Leu 50 55 60 TCT ATT AGT GGT AGA GAT GCA GTT CAG ACT GCG CTT ACT GTTGTT GGG 901 Ser Ile Ser Gly Arg Asp Ala Val Gln Thr Ala Leu Thr Val ValGly 65 70 75 AGA ATA CTC GGG GCT TTA GGT GTT CCG TTT TCT GGA CAA ATA GTGAGT 949 Arg Ile Leu Gly Ala Leu Gly Val Pro Phe Ser Gly Gln Ile Val Ser80 85 90 TTT TAT CAA TTC CTT TTA AAT ACA CTG TGG CCA GTT AAT GAT ACA GCT997 Phe Tyr Gln Phe Leu Leu Asn Thr Leu Trp Pro Val Asn Asp Thr Ala 95100 105 110 ATA TGG GAA GCT TTC ATG CGA CAG GTG GAG GAA CTT GTC AAT CAACAA 1045 Ile Trp Glu Ala Phe Met Arg Gln Val Glu Glu Leu Val Asn Gln Gln115 120 125 ATA ACA GAA TTT GCA AGA AAT CAG GCA CTT GCA AGA TTG CAA GGATTA 1093 Ile Thr Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly Leu130 135 140 GGA GAC TCT TTT AAT GTA TAT CAA CGT TCC CTT CAA AAT TGG TTGGCT 1141 Gly Asp Ser Phe Asn Val Tyr Gln Arg Ser Leu Gln Asn Trp Leu Ala145 150 155 GAT CGA AAT GAT ACA CGA AAT TTA AGT GTT GTT CGT GCT CAA TTTATA 1189 Asp Arg Asn Asp Thr Arg Asn Leu Ser Val Val Arg Ala Gln Phe Ile160 165 170 GCT TTA GAC CTT GAT TTT GTT AAT GCT ATT CCA TTG TTT GCA GTAAAT 1237 Ala Leu Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Phe Ala Val Asn175 180 185 190 GGA CAG CAG GTT CCA TTA CTG TCA GTA TAT GCA CAA GCT GTGAAT TTA 1285 Gly Gln Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Val AsnLeu 195 200 205 CAT TTG TTA TTA TTA AAA GAT GCA TCT CTT TTT GGA GAA GGATGG GGA 1333 His Leu Leu Leu Leu Lys Asp Ala Ser Leu Phe Gly Glu Gly TrpGly 210 215 220 TTC ACA CAG GGG GAA ATT TCC ACA TAT TAT GAC CGT CAA TTGGAA CTA 1381 Phe Thr Gln Gly Glu Ile Ser Thr Tyr Tyr Asp Arg Gln Leu GluLeu 225 230 235 ACC GCT AAG TAC ACT AAT TAC TGT GAA ACT TGG TAT AAT ACAGGT TTA 1429 Thr Ala Lys Tyr Thr Asn Tyr Cys Glu Thr Trp Tyr Asn Thr GlyLeu 240 245 250 GAT CGT TTA AGA GGA ACA AAT ACT GAA AGT TGG TTA AGA TATCAT CAA 1477 Asp Arg Leu Arg Gly Thr Asn Thr Glu Ser Trp Leu Arg Tyr HisGln 255 260 265 270 TTC CGT AGA GAA ATG ACT TTA GTG GTA TTA GAT GTT GTGGCG CTA TTT 1525 Phe Arg Arg Glu Met Thr Leu Val Val Leu Asp Val Val AlaLeu Phe 275 280 285 CCA TAT TAT GAT GTA CGA CTT TAT CCA ACG GGA TCA AACCCA CAG CTT 1573 Pro Tyr Tyr Asp Val Arg Leu Tyr Pro Thr Gly Ser Asn ProGln Leu 290 295 300 ACA CGT GAG GTA TAT ACA GAT CCG ATT GTA TTT AAT CCACCA GCT AAT 1621 Thr Arg Glu Val Tyr Thr Asp Pro Ile Val Phe Asn Pro ProAla Asn 305 310 315 GTT GGA CTT TGC CGA CGT TGG GGT ACT AAT CCC TAT AATACT TTT TCT 1669 Val Gly Leu Cys Arg Arg Trp Gly Thr Asn Pro Tyr Asn ThrPhe Ser 320 325 330 GAG CTC GAA AAT GCC TTC ATT CGC CCA CCA CAT CTT TTTGAT AGG CTG 1717 Glu Leu Glu Asn Ala Phe Ile Arg Pro Pro His Leu Phe AspArg Leu 335 340 345 350 AAT AGC TTA ACA ATC AGC AGT AAT CGA TTT CCA GTTTCA TCT AAT TTT 1765 Asn Ser Leu Thr Ile Ser Ser Asn Arg Phe Pro Val SerSer Asn Phe 355 360 365 ATG GAT TAT TGG TCA GGA CAT ACG TTA CGC CGT AGTTAT CTG AAC GAT 1813 Met Asp Tyr Trp Ser Gly His Thr Leu Arg Arg Ser TyrLeu Asn Asp 370 375 380 TCA GCA GTA CAA GAA GAT AGT TAT GGC CTA ATT ACAACC ACA AGA GCA 1861 Ser Ala Val Gln Glu Asp Ser Tyr Gly Leu Ile Thr ThrThr Arg Ala 385 390 395 ACA ATT AAT CCC GGA GTT GAT GGA ACA AAC CGC ATAGAG TCA ACG GCA 1909 Thr Ile Asn Pro Gly Val Asp Gly Thr Asn Arg Ile GluSer Thr Ala 400 405 410 GTA GAT TTT CGT TCT GCA TTG ATA GGT ATA TAT GGCGTG AAT AGA GCT 1957 Val Asp Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly ValAsn Arg Ala 415 420 425 430 TCT TTT GTC CCA GGA GGC TTG TTT AAT GGT ACGACT TCT CCT GCT AAT 2005 Ser Phe Val Pro Gly Gly Leu Phe Asn Gly Thr ThrSer Pro Ala Asn 435 440 445 GGA GGA TGT AGA GAT CTC TAT GAT ACA AAT GATGAA TTA CCA CCA GAT 2053 Gly Gly Cys Arg Asp Leu Tyr Asp Thr Asn Asp GluLeu Pro Pro Asp 450 455 460 GAA AGT ACC GGA AGT TCA ACC CAT AGA CTA TCTCAT GTT ACC TTT TTT 2101 Glu Ser Thr Gly Ser Ser Thr His Arg Leu Ser HisVal Thr Phe Phe 465 470 475 AGC TTT CAA ACT AAT CAG GCT GGA TCT ATA GCTAAT GCA GGA AGT GTA 2149 Ser Phe Gln Thr Asn Gln Ala Gly Ser Ile Ala AsnAla Gly Ser Val 480 485 490 CCT ACT TAT GTT TGG ACC CGT CGT GAT GTG GACCTT AAT AAT ACG ATT 2197 Pro Thr Tyr Val Trp Thr Arg Arg Asp Val Asp LeuAsn Asn Thr Ile 495 500 505 510 ACC CCA AAT AGA ATT ACA CAA TTA CCA TTGGTA AAG GCA TCT GCA CCT 2245 Thr Pro Asn Arg Ile Thr Gln Leu Pro Leu ValLys Ala Ser Ala Pro 515 520 525 GTT TCG GGT ACT ACG GTC TTA AAA GGT CCAGGA TTT ACA GGA GGG GGT 2293 Val Ser Gly Thr Thr Val Leu Lys Gly Pro GlyPhe Thr Gly Gly Gly 530 535 540 ATA CTC CGA AGA ACA ACT AAT GGC ACA TTTGGA ACG TTA AGA GTA ACG 2341 Ile Leu Arg Arg Thr Thr Asn Gly Thr Phe GlyThr Leu Arg Val Thr 545 550 555 GTT AAT TCA CCA TTA ACA CAA CAA TAT CGCCTA AGA GTT CGT TTT GCC 2389 Val Asn Ser Pro Leu Thr Gln Gln Tyr Arg LeuArg Val Arg Phe Ala 560 565 570 TCA ACA GGA AAT TTC AGT ATA AGG GTA CTCCGT GGA GGG GTT TCT ATC 2437 Ser Thr Gly Asn Phe Ser Ile Arg Val Leu ArgGly Gly Val Ser Ile 575 580 585 590 GGT GAT GTT AGA TTA GGG AGC ACA ATGAAC AGA GGG CAG GAA CTA ACT 2485 Gly Asp Val Arg Leu Gly Ser Thr Met AsnArg Gly Gln Glu Leu Thr 595 600 605 TAC GAA TCC TTT TTC ACA AGA GAG TTTACT ACT ACT GGT CCG TTC AAT 2533 Tyr Glu Ser Phe Phe Thr Arg Glu Phe ThrThr Thr Gly Pro Phe Asn 610 615 620 CCG CCT TTT ACA TTT ACA CAA GCT CAAGAG ATT CTA ACA GTG AAT GCA 2581 Pro Pro Phe Thr Phe Thr Gln Ala Gln GluIle Leu Thr Val Asn Ala 625 630 635 GAA GGT GTT AGC ACC GGT GGT GAA TATTAT ATA GAT AGA ATT GAA ATT 2629 Glu Gly Val Ser Thr Gly Gly Glu Tyr TyrIle Asp Arg Ile Glu Ile 640 645 650 GTC CCT GTG AAT CCG GCA CGA GAA GCGGAA GAG GAT TTA GAA GCG GCG 2677 Val Pro Val Asn Pro Ala Arg Glu Ala GluGlu Asp Leu Glu Ala Ala 655 660 665 670 AAG AAA GCG GTG GCG AGC TTG TTTACA CGT ACA AGG GAC GGA TTA CAG 2725 Lys Lys Ala Val Ala Ser Leu Phe ThrArg Thr Arg Asp Gly Leu Gln 675 680 685 GTA AAT GTG ACA GAT TAT CAA GTGGAC CAA GCG GCA AAT TTA GTG TCA 2773 Val Asn Val Thr Asp Tyr Gln Val AspGln Ala Ala Asn Leu Val Ser 690 695 700 TGC TTA TCC GAT GAA CAA TAT GGGCAT GAC AAA AAG ATG TTA TTG GAA 2821 Cys Leu Ser Asp Glu Gln Tyr Gly HisAsp Lys Lys Met Leu Leu Glu 705 710 715 GCG GTA AGA GCG GCA AAA CGC CTCAGC CGC GAA CGC AAC TTA CTT CAA 2869 Ala Val Arg Ala Ala Lys Arg Leu SerArg Glu Arg Asn Leu Leu Gln 720 725 730 GAT CCA GAT TTT AAT ACA ATC AATAGT ACA GAA GAG AAT GGC TGG AAG 2917 Asp Pro Asp Phe Asn Thr Ile Asn SerThr Glu Glu Asn Gly Trp Lys 735 740 745 750 GCA AGT AAC GGT GTT ACT ATTAGC GAG GGC GGT CCA TTC TTT AAA GGT 2965 Ala Ser Asn Gly Val Thr Ile SerGlu Gly Gly Pro Phe Phe Lys Gly 755 760 765 CGT GCA CTT CAG TTA GCA AGCGCA AGA GAA AAT TAT CCA ACA TAC ATT 3013 Arg Ala Leu Gln Leu Ala Ser AlaArg Glu Asn Tyr Pro Thr Tyr Ile 770 775 780 TAT CAA AAA GTA GAT GCA TCGGTG TTA AAG CCT TAT ACA CGC TAT AGA 3061 Tyr Gln Lys Val Asp Ala Ser ValLeu Lys Pro Tyr Thr Arg Tyr Arg 785 790 795 CTA GAT GGA TTT GTG AAG AGTAGT CAA GAT TTA GAA ATT GAT CTC ATC 3109 Leu Asp Gly Phe Val Lys Ser SerGln Asp Leu Glu Ile Asp Leu Ile 800 805 810 CAC CAT CAT AAA GTC CAT CTTGTA AAA AAT GTA CCA GAT AAT TTA GTA 3157 His His His Lys Val His Leu ValLys Asn Val Pro Asp Asn Leu Val 815 820 825 830 TCT GAT ACT TAC TCA GATGGT TCT TGC AGC GGA ATC AAC CGT TGT GAT 3205 Ser Asp Thr Tyr Ser Asp GlySer Cys Ser Gly Ile Asn Arg Cys Asp 835 840 845 GAA CAG CAT CAG GTA GATATG CAG CTA GAT GCG GAG CAT CAT CCA ATG 3253 Glu Gln His Gln Val Asp MetGln Leu Asp Ala Glu His His Pro Met 850 855 860 GAT TGC TGT GAA GCG GCTCAA ACA CAT GAG TTT TCT TCC TAT ATT AAT 3301 Asp Cys Cys Glu Ala Ala GlnThr His Glu Phe Ser Ser Tyr Ile Asn 865 870 875 ACA GGG GAT CTA AAT GCAAGT GTA GAT CAG GGC ATT TGG GTT GTA TTA 3349 Thr Gly Asp Leu Asn Ala SerVal Asp Gln Gly Ile Trp Val Val Leu 880 885 890 AAA GTT CGA ACA ACA GATGGG TAT GCG ACG TTA GGA AAT CTT GAA TTG 3397 Lys Val Arg Thr Thr Asp GlyTyr Ala Thr Leu Gly Asn Leu Glu Leu 895 900 905 910 GTA GAG GTT GGG CCATTA TCG GGT GAA TCT CTA GAA CGG GAA CAA AGA 3445 Val Glu Val Gly Pro LeuSer Gly Glu Ser Leu Glu Arg Glu Gln Arg 915 920 925 GAT AAT GCG AAA TGGAAT GCA GAG CTA GGA AGA AAA CGT GCA GAA ATA 3493 Asp Asn Ala Lys Trp AsnAla Glu Leu Gly Arg Lys Arg Ala Glu Ile 930 935 940 GAT CGT GTG TAT TTAGCT GCG AAA CAA GCA ATT AAT CAT CTG TTT GTA 3541 Asp Arg Val Tyr Leu AlaAla Lys Gln Ala Ile Asn His Leu Phe Val 945 950 955 GAC TAT CAA GAT CAACAA TTA AAT CCA GAA ATT GGG CTA GCA GAA ATT 3589 Asp Tyr Gln Asp Gln GlnLeu Asn Pro Glu Ile Gly Leu Ala Glu Ile 960 965 970 AAT GAA GCT TCA AATCTT GTA GAG TCA ATT TCG GGT GTA TAT AGT GAT 3637 Asn Glu Ala Ser Asn LeuVal Glu Ser Ile Ser Gly Val Tyr Ser Asp 975 980 985 990 ACA CTA TTA CAGATT CCT GGG ATT AAC TAC GAA ATT TAC ACA GAG TTA 3685 Thr Leu Leu Gln IlePro Gly Ile Asn Tyr Glu Ile Tyr Thr Glu Leu 995 1000 1005 TCC GAT CGCTTA CAA CAA GCA TCG TAT CTG TAT ACG TCT AGA AAT GCG 3733 Ser Asp Arg LeuGln Gln Ala Ser Tyr Leu Tyr Thr Ser Arg Asn Ala 1010 1015 1020 GTG CAAAAT GGA GAC TTT AAC AGT GGT CTA GAT AGT TGG AAT ACA ACT 3781 Val Gln AsnGly Asp Phe Asn Ser Gly Leu Asp Ser Trp Asn Thr Thr 1025 1030 1035 ATGGAT GCA TCG GTT CAG CAA GAT GGC AAT ATG CAT TTC TTA GTT CTT 3829 Met AspAla Ser Val Gln Gln Asp Gly Asn Met His Phe Leu Val Leu 1040 1045 1050TCG CAT TGG GAT GCA CAA GTT TCC CAA CAA TTG AGA GTA AAT CCG AAT 3877 SerHis Trp Asp Ala Gln Val Ser Gln Gln Leu Arg Val Asn Pro Asn 1055 10601065 1070 TGT AAG TAT GTC TTA CGT GTG ACA GCA AGA AAA GTA GGA GGC GGAGAT 3925 Cys Lys Tyr Val Leu Arg Val Thr Ala Arg Lys Val Gly Gly Gly Asp1075 1080 1085 GGA TAC GTC ACA ATC CGA GAT GGC GCT CAT CAC CAA GAA ACTCTT ACA 3973 Gly Tyr Val Thr Ile Arg Asp Gly Ala His His Gln Glu Thr LeuThr 1090 1095 1100 TTT AAT GCA TGT GAC TAC GAT GTA AAT GGT ACG TAT GTCAAT GAC AAT 4021 Phe Asn Ala Cys Asp Tyr Asp Val Asn Gly Thr Tyr Val AsnAsp Asn 1105 1110 1115 TCG TAT ATA ACA GAA GAA GTG GTA TTC TAC CCA GAGACA AAA CAT ATG 4069 Ser Tyr Ile Thr Glu Glu Val Val Phe Tyr Pro Glu ThrLys His Met 1120 1125 1130 TGG GTA GAG GTG AGT GAA TCC GAA GGT TCA TTCTAT ATA GAC AGT ATT 4117 Trp Val Glu Val Ser Glu Ser Glu Gly Ser Phe TyrIle Asp Ser Ile 1135 1140 1145 1150 GAG TTT ATT GAA ACA CAA GAG TAGAAGAGGGGGA TCCTAACGTA TAGCAACTAT 4171 Glu Phe Ile Glu Thr Gln Glu * 1155GAGAGGATAC TCCGTACAAA CAAAGATTAA AAAAAGGTAA AATGAATAGA ACCCCCTACT 4231GGTAGAAGGA CCGATAGGGG GTTCTTACAT GAAAAAATGT AGCTGTTTAC TAAGGTGTAT 4291AAAAAACAGC ATATCTGATA GAAAAAAGTG AGTACCTTAT AAAGAAAGAA TTC 4344 (2)INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH:1157 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Met Asn Arg AsnAsn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His 1 5 10 15 Cys Gly CysPro Ser Asp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp 20 25 30 Pro Asn AlaAla Leu Gln Asn Met Asn Tyr Lys Asp Tyr Leu Gln Met 35 40 45 Thr Asp GluAsp Tyr Thr Asp Ser Tyr Ile Asn Pro Ser Leu Ser Ile 50 55 60 Ser Gly ArgAsp Ala Val Gln Thr Ala Leu Thr Val Val Gly Arg Ile 65 70 75 80 Leu GlyAla Leu Gly Val Pro Phe Ser Gly Gln Ile Val Ser Phe Tyr 85 90 95 Gln PheLeu Leu Asn Thr Leu Trp Pro Val Asn Asp Thr Ala Ile Trp 100 105 110 GluAla Phe Met Arg Gln Val Glu Glu Leu Val Asn Gln Gln Ile Thr 115 120 125Glu Phe Ala Arg Asn Gln Ala Leu Ala Arg Leu Gln Gly Leu Gly Asp 130 135140 Ser Phe Asn Val Tyr Gln Arg Ser Leu Gln Asn Trp Leu Ala Asp Arg 145150 155 160 Asn Asp Thr Arg Asn Leu Ser Val Val Arg Ala Gln Phe Ile AlaLeu 165 170 175 Asp Leu Asp Phe Val Asn Ala Ile Pro Leu Phe Ala Val AsnGly Gln 180 185 190 Gln Val Pro Leu Leu Ser Val Tyr Ala Gln Ala Val AsnLeu His Leu 195 200 205 Leu Leu Leu Lys Asp Ala Ser Leu Phe Gly Glu GlyTrp Gly Phe Thr 210 215 220 Gln Gly Glu Ile Ser Thr Tyr Tyr Asp Arg GlnLeu Glu Leu Thr Ala 225 230 235 240 Lys Tyr Thr Asn Tyr Cys Glu Thr TrpTyr Asn Thr Gly Leu Asp Arg 245 250 255 Leu Arg Gly Thr Asn Thr Glu SerTrp Leu Arg Tyr His Gln Phe Arg 260 265 270 Arg Glu Met Thr Leu Val ValLeu Asp Val Val Ala Leu Phe Pro Tyr 275 280 285 Tyr Asp Val Arg Leu TyrPro Thr Gly Ser Asn Pro Gln Leu Thr Arg 290 295 300 Glu Val Tyr Thr AspPro Ile Val Phe Asn Pro Pro Ala Asn Val Gly 305 310 315 320 Leu Cys ArgArg Trp Gly Thr Asn Pro Tyr Asn Thr Phe Ser Glu Leu 325 330 335 Glu AsnAla Phe Ile Arg Pro Pro His Leu Phe Asp Arg Leu Asn Ser 340 345 350 LeuThr Ile Ser Ser Asn Arg Phe Pro Val Ser Ser Asn Phe Met Asp 355 360 365Tyr Trp Ser Gly His Thr Leu Arg Arg Ser Tyr Leu Asn Asp Ser Ala 370 375380 Val Gln Glu Asp Ser Tyr Gly Leu Ile Thr Thr Thr Arg Ala Thr Ile 385390 395 400 Asn Pro Gly Val Asp Gly Thr Asn Arg Ile Glu Ser Thr Ala ValAsp 405 410 415 Phe Arg Ser Ala Leu Ile Gly Ile Tyr Gly Val Asn Arg AlaSer Phe 420 425 430 Val Pro Gly Gly Leu Phe Asn Gly Thr Thr Ser Pro AlaAsn Gly Gly 435 440 445 Cys Arg Asp Leu Tyr Asp Thr Asn Asp Glu Leu ProPro Asp Glu Ser 450 455 460 Thr Gly Ser Ser Thr His Arg Leu Ser His ValThr Phe Phe Ser Phe 465 470 475 480 Gln Thr Asn Gln Ala Gly Ser Ile AlaAsn Ala Gly Ser Val Pro Thr 485 490 495 Tyr Val Trp Thr Arg Arg Asp ValAsp Leu Asn Asn Thr Ile Thr Pro 500 505 510 Asn Arg Ile Thr Gln Leu ProLeu Val Lys Ala Ser Ala Pro Val Ser 515 520 525 Gly Thr Thr Val Leu LysGly Pro Gly Phe Thr Gly Gly Gly Ile Leu 530 535 540 Arg Arg Thr Thr AsnGly Thr Phe Gly Thr Leu Arg Val Thr Val Asn 545 550 555 560 Ser Pro LeuThr Gln Gln Tyr Arg Leu Arg Val Arg Phe Ala Ser Thr 565 570 575 Gly AsnPhe Ser Ile Arg Val Leu Arg Gly Gly Val Ser Ile Gly Asp 580 585 590 ValArg Leu Gly Ser Thr Met Asn Arg Gly Gln Glu Leu Thr Tyr Glu 595 600 605Ser Phe Phe Thr Arg Glu Phe Thr Thr Thr Gly Pro Phe Asn Pro Pro 610 615620 Phe Thr Phe Thr Gln Ala Gln Glu Ile Leu Thr Val Asn Ala Glu Gly 625630 635 640 Val Ser Thr Gly Gly Glu Tyr Tyr Ile Asp Arg Ile Glu Ile ValPro 645 650 655 Val Asn Pro Ala Arg Glu Ala Glu Glu Asp Leu Glu Ala AlaLys Lys 660 665 670 Ala Val Ala Ser Leu Phe Thr Arg Thr Arg Asp Gly LeuGln Val Asn 675 680 685 Val Thr Asp Tyr Gln Val Asp Gln Ala Ala Asn LeuVal Ser Cys Leu 690 695 700 Ser Asp Glu Gln Tyr Gly His Asp Lys Lys MetLeu Leu Glu Ala Val 705 710 715 720 Arg Ala Ala Lys Arg Leu Ser Arg GluArg Asn Leu Leu Gln Asp Pro 725 730 735 Asp Phe Asn Thr Ile Asn Ser ThrGlu Glu Asn Gly Trp Lys Ala Ser 740 745 750 Asn Gly Val Thr Ile Ser GluGly Gly Pro Phe Phe Lys Gly Arg Ala 755 760 765 Leu Gln Leu Ala Ser AlaArg Glu Asn Tyr Pro Thr Tyr Ile Tyr Gln 770 775 780 Lys Val Asp Ala SerVal Leu Lys Pro Tyr Thr Arg Tyr Arg Leu Asp 785 790 795 800 Gly Phe ValLys Ser Ser Gln Asp Leu Glu Ile Asp Leu Ile His His 805 810 815 His LysVal His Leu Val Lys Asn Val Pro Asp Asn Leu Val Ser Asp 820 825 830 ThrTyr Ser Asp Gly Ser Cys Ser Gly Ile Asn Arg Cys Asp Glu Gln 835 840 845His Gln Val Asp Met Gln Leu Asp Ala Glu His His Pro Met Asp Cys 850 855860 Cys Glu Ala Ala Gln Thr His Glu Phe Ser Ser Tyr Ile Asn Thr Gly 865870 875 880 Asp Leu Asn Ala Ser Val Asp Gln Gly Ile Trp Val Val Leu LysVal 885 890 895 Arg Thr Thr Asp Gly Tyr Ala Thr Leu Gly Asn Leu Glu LeuVal Glu 900 905 910 Val Gly Pro Leu Ser Gly Glu Ser Leu Glu Arg Glu GlnArg Asp Asn 915 920 925 Ala Lys Trp Asn Ala Glu Leu Gly Arg Lys Arg AlaGlu Ile Asp Arg 930 935 940 Val Tyr Leu Ala Ala Lys Gln Ala Ile Asn HisLeu Phe Val Asp Tyr 945 950 955 960 Gln Asp Gln Gln Leu Asn Pro Glu IleGly Leu Ala Glu Ile Asn Glu 965 970 975 Ala Ser Asn Leu Val Glu Ser IleSer Gly Val Tyr Ser Asp Thr Leu 980 985 990 Leu Gln Ile Pro Gly Ile AsnTyr Glu Ile Tyr Thr Glu Leu Ser Asp 995 1000 1005 Arg Leu Gln Gln AlaSer Tyr Leu Tyr Thr Ser Arg Asn Ala Val Gln 1010 1015 1020 Asn Gly AspPhe Asn Ser Gly Leu Asp Ser Trp Asn Thr Thr Met Asp 1025 1030 1035 1040Ala Ser Val Gln Gln Asp Gly Asn Met His Phe Leu Val Leu Ser His 10451050 1055 Trp Asp Ala Gln Val Ser Gln Gln Leu Arg Val Asn Pro Asn CysLys 1060 1065 1070 Tyr Val Leu Arg Val Thr Ala Arg Lys Val Gly Gly GlyAsp Gly Tyr 1075 1080 1085 Val Thr Ile Arg Asp Gly Ala His His Gln GluThr Leu Thr Phe Asn 1090 1095 1100 Ala Cys Asp Tyr Asp Val Asn Gly ThrTyr Val Asn Asp Asn Ser Tyr 1105 1110 1115 1120 Ile Thr Glu Glu Val ValPhe Tyr Pro Glu Thr Lys His Met Trp Val 1125 1130 1135 Glu Val Ser GluSer Glu Gly Ser Phe Tyr Ile Asp Ser Ile Glu Phe 1140 1145 1150 Ile GluThr Gln Glu 1155

What is claimed is:
 1. An isolated protein variant of the protein of SEQID NO:4, which variant has the same insecticidal characteristics as theprotein of SEQ ID NO:4 or an insecticidally effective fragment thereof,and which variant comprises an amino acid sequence which is encoded by aDNA sequence hybridizing under stringent hybridization conditions to theDNA and wherein said stringent hybridization conditions are establishedas follows using the following consecutive steps: a) immobilizing DNAfragments on a filter; b) prehybridizing said filter for 1 to 2 hours at42° C. in 50% formamide, 5×SSPE, 2×Denhardt's reagent and 0.1% SDS, orfor 1 to 2 hours at 68° C. in 6×SSC, 2×Denhardt's reagent and 0.1% SDS;c) adding a hybridization probe which has been radiolabeled; d)incubating for 16 to 24 hours; e) washing said filter for 20 minutes atroom temperature in 1×SSC, 0.1% SDS; and f) washing said filter threetimes for 20 minutes each at 68° C. in 0.2×SSC, 0.1% SDS; and g)autoradiographing said filter by exposing said filter for 24 to 48 hoursto X-ray film at −70° C. with an intensifying screen.
 2. The protein ofclaim 1 which is expressed in a transformed plant or a transformedmicroorganism.
 3. The protein of claim 1, wherein said protein varianthas the same insecticidal characteristics as the protein of SEQ ID NO:4or an insecticidally-effective fragment thereof, against an insectselected from the group consisting of Spodoptera exigua, Spodopteralittoralis, Spodoptera frugiperda, Agrotis ipsilon, Mamestra brassica,Heliothis virescens, Ostrinia nubilalis, and Plutella xylostella.
 4. Anisolated protein variant of the protein of SEQ ID NO:4, which varianthas the same insecticidal characteristics as the protein of SEQ ID NO:4or an insecticidally effective fragment thereof, and which variantcomprises an amino acid sequence which is encoded by a DNA sequencehybridizing under stringent hybridization conditions to the DNA of SEQID NO:4 from nucleotide position 797 to nucleotide position 2641 andwherein said stringent hybridization conditions are established asfollows using the following consecutive steps: a) immobilizing DNAfragments on a filter; b) prehybridizing said filter for 1 to 2 hours at42° C. in 50% formamide, 5×SSPE, 2×Denhardt's reagent and 0.1% SDS, orfor 1 to 2 hours at 68° C. in 6×SSC, 2×Denhardt's reagent and 0.1% SDS;c) adding a hybridization probe which has been radiolabeled; d)incubating for 16 to 24 hours; e) washing said filter for 20 minutes atroom temperature in 1×SSC, 0.1% SDS; and f) washing said filter threetimes for 20 minutes each at 68° C. in 0.2×SSC, 0.1% SDS; and g)autoradiographing said filter by exposing said filter for to 48 hours toX-ray film at −70° C. with an intensifying screen.
 5. The protein ofclaim 4, wherein said protein variant has the same insecticidalcharacteristics as the protein of SEQ ID NO:4 or aninsecticidally-effective fragment thereof, against an insect selectedfrom the group consisting of Spodoptera exigua, Spodoptera littoralis,Spodoptera frugiperda, Agrotis ipsilon, Mamestra brassica, Heliothisvirescens, Ostrinia nubilalis, and Plutella xylostella.
 6. An isolatedDNA encoding the protein variant of claim
 1. 7. The DNA of claim 6,wherein said DNA is an artificial sequence with a modified codon usage.8. A plant cell or plant transformed to contain a chimeric genecomprising as coding sequence the DNA of claim
 6. 9. A microorganism,transformed to contain a chimeric gene comprising as coding sequence theDNA of claim
 6. 10. An isolated DNA comprising a coding regioncomprising the nucleotide sequence of SEQ ID NO:4 or a fragment thereofencoding an insecticidally effective protein portion, or a degeneratenucleotide sequence thereof wherein one or more amino acid codons havebeen replaced with others without changing the amino acid sequence ofthe protein or its insecticidally effective portion.